Probiotics’ Effects in the Treatment of Anxiety and Depression: A Comprehensive Review of 2014–2023 Clinical Trials

Changes in the gut microbiome can affect cognitive and psychological functions via the microbiota–gut–brain (MGB) axis. Probiotic supplements are thought to have largely positive effects on mental health when taken in sufficient amounts; however, despite extensive research having been conducted, there is a lack of consistent findings on the effects of probiotics on anxiety and depression and the associated microbiome alterations. The aim of our study is to systematically review the most recent literature of the last 10 years in order to clarify whether probiotics could actually improve depression and anxiety symptoms. Our results indicate that the majority of the most recent literature suggests a beneficial role of probiotics in the treatment of depression and anxiety, despite the existence of a substantial number of less positive findings. Given probiotics’ potential to offer novel, personalized treatment options for mood disorders, further, better targeted research in psychiatric populations is needed to address concerns about the exact mechanisms of probiotics, dosing, timing of treatment, and possible differences in outcomes depending on the severity of anxiety and depression.


Introduction
Several microbial environments exist in different locations inside the human body, such as the skin, nasal mucosa, and gastrointestinal tract, consisting of a large number of bacteria, viruses, fungi, and protozoa, referred to as the microbiota [1].Thousands of different microbe species have been identified in human beings, with most of them classified in the Proteobacteria, Firmicutes, Actinobacteria, and Bacteroidetes phyla [2].The microbiome (i.e., the entire environment, including the microorganisms, their genomes, and the surrounding environmental conditions) [3] of the gut contains the largest and most diverse population of microorganisms, that are critical for a variety of physiological functions, including preserving gut integrity or forming the intestinal epithelium [4], obtaining energy [5], protecting against infections [6], and controlling host immunity [7]; it is thus considered an "invisible organ" of the human body [8].
The gut-brain axis is a system which encompasses a bidirectional communication between the gut and the brain [9] involving the neural (vagus nerve and enteric nervous system), immune (cytokines), endocrine (cortisol), and metabolic (short-chain fatty acids) pathways [10,11].Gut-produced cytokines can reach the brain through the bloodstream and although it is doubtful that they would cross the blood-brain barrier (BBB) under normal physiological conditions, there is growing evidence that they can affect parts of the brain where the BBB is inadequate, such as the hypothalamus; for instance, cytokines interleukin-1 (IL-1) and IL-6 trigger the release of cortisol by activating the hypothalamicpituitary-adrenal (HPA) axis [12].In addition, gut microbiota is the primary source of short-chain fatty acids (SCFAs), which modulate brain health and behavior via the immune system, having a beneficial anti-inflammatory and mental health role by inducing T cell differentiation, controlling inflammatory cytokine production, and influencing serotonin and other neurotransmitter production [13].The gut microbiome is also able to metabolize glutamate to produce certain beneficial metabolites, such as GABA and serotonin, which are known molecules thought to reduce anxious and depressive states [14]; despite not being able to cross the blood-brain barrier (BBB), they can nevertheless pass through the intestinal mucosal layer and have an indirect effect on brain function via the enteric nervous system [15].As a result, the gut microbiome plays a central role in modulating the reciprocal communication between the gut and brain and together they comprise the microbiota-gut-brain (MGB) axis [3].Increasing evidence suggests that the gut microbiome and the MGB axis play a crucial role in modulating psychiatric disorders [16].Therefore, treatments such as probiotics that potentially alter the composition and function of the gut microbiome [17], might have desired beneficial effects in human mental health [18].
Probiotics are described as "living microorganisms" that when administered in adequate amounts confer health benefits on the host [19]; psychobiotics are defined as those probiotics that can specifically bestow mental health benefits.This idea has been extended to include "prebiotics", that promote the proliferation of beneficial bacteria in the gastrointestinal tract [20].The combination of probiotics and prebiotics is often described as synbiotics; however, the term has been recently redefined as "a mixture comprising live microorganisms and substrate(s) selectively utilized by host microorganisms that confers a health benefit on the host" [21].Existing evidence suggests that probiotics may help patients with conditions such as IBS and persistent exhaustion feel emotionally better and live with less stress [22], while certain elements of brain function and behavior, particularly those that are vagus nerve dependent, can be altered by probiotic strains [23].The mechanisms of action of probiotics are diverse, heterogeneous, and strain specific [24].The beneficial effects of probiotics have largely been attributed to the modification of the intestinal microbiota, through improving the functioning of the microbial populations already present and thus limiting pathogens [25].It is also accomplished through the colonization and normalization of disrupted intestinal microbial communities in humans, alongside the competitive exclusion of pathogens and the production of bacteriocins [24].Moreover, probiotics have been found to immunomodulate the body, promote the growth and differentiation of epithelial cells, reinforce the intestinal barrier [26], and create antimicrobial substances or metabolic products that inhibit the growth of other possibly harmful bacteria [17], while also fighting with them for receptors and binding points on the intestinal mucosa [27], preserving the gut microbiome's harmony [28].
The effect of probiotics in anxiety and depression is currently being extensively investigated given their potential to be novel, adverse event-free, and customizable treatments [18].Clinical trials in humans have overall shown that probiotic and prebiotic therapies have beneficial mental health effects.For instance, the probiotic combination of L. helveticus and B. longum, when given to healthy human volunteers for one month, has been shown to reduce psychological distress [29], while patients with IBS who received high intake of prebiotic trans-GOS for 12 weeks reported significantly improved subjective global assessment scores and anxiety scores compared to placebo treatment [30].Moreover, participants in a healthy state who consumed a milk drink containing probiotics on a daily basis presented with a considerable boost in their mood after three weeks when compared to a placebo [31].
When looking at meta-analytic data, two relatively older studies from 2016 and 2017 examining the effectiveness of probiotics in alleviating depression revealed a general improvement of depressive symptoms following probiotic ingestion, with minimal potential for negative side effects [32,33].A more recent meta-analysis supported the use of probiotics in depressed patients with or without concomitant somatic disorders; however, it could not draw firm conclusions on the effectiveness of probiotics in the healthy population [34].On the contrary, another meta-analysis showed that probiotics can reduce subjective stress level in healthy volunteers [35].A 2021 meta-analysis demonstrated with moderate-and low-certainty evidence that probiotics improve symptoms of depression and anxiety in clinical patients measured with the Beck Depression Index and the State-Trait Anxiety Inventory but not on any other scales [36].
However, other meta-analytic data have been less positive.A recent study demonstrated that most probiotics did not affect mood, stress, anxiety, depression, and psychiatric distress when compared to placebo at the qualitative level, indicating that there is not yet strong enough evidence to support the inclusion of probiotic, prebiotic and symbiotic supplements in treatment guidelines for depression [37].Another meta-analysis suggested that the current evidence base for prebiotics and probiotics in the treatment of internalizing disorders appears modest [38], while a 2020 meta-analysis reported that not all probiotic treatments exhibited a psychobiotic effect on the central nervous system (CNS) [39].Moreover, another study found no significant difference between the probiotics and placebo groups in alleviating anxiety and suggested that probiotics should be used in clinical subjects or the general population only when more valid evidence in this area is obtained [40].
Therefore, despite the vast scale of research that has been taking place in the multibillion dollar probiotic industry [18] in the last years there is a lack of consensus of the existing evidence on how to best use them in mental health, which might cause confusion in healthcare professionals [41].Thus, and given the increasingly broader range of probiotic products used [41] and the rapidly increasing literature in the probiotic field [42], the aim of our study was to provide a comprehensive update of the last decade research data on the effectiveness of probiotics in the treatment of anxiety and depression.Taking into consideration the huge potential of probiotics to become novel, personalized treatments for mental health disorders, the academic importance of our study lies in the effort to analyze the most recent (last ten years) accumulated knowledge in the field and shed more light on the question of whether and to what degree probiotics are actually effective in depression and anxiety treatment.

Materials and Methods
The Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) checklist was used to guide this study.A protocol has not been registered; however, our study methods were designed and formulated a priori.

Search Strategy
Two databases (MEDLINE and Scopus) were selected to carry out the present literature search, which was conducted by two investigators (TM, DM).To trace all relevant studies published between 1 January 2014 and 31 December 2023, the following keywords were used: "probiotics" OR "prebiotics" OR "psychobiotics" AND "gut microbiota" OR "gut microbiome" AND "depression" OR "anxiety".All retrieved articles were also hand searched for any further potential eligible articles.Any disagreement regarding the screening or selection process was solved by a third investigator (EM) until a consensus was reached.

Selection Criteria
Only full-text original research articles (randomized controlled trials and clinical trials) published in the English language were included.Secondary analyses, reviews, guidelines, notes, errata, letters, meeting summaries, comments, unpublished abstracts, retracted articles, or studies conducted on animals or children (<18 years old) were excluded.There was no restriction on study design or other sample characteristics.

Data Extraction
Data extraction was performed independently by two investigators from the team (DT and KT) using a predefined data form created in Excel.The same two investigators also rated the quality of the included studies using the Jadad scale [43], also known as the Oxford quality scoring system, in order to evaluate the methodological quality of clinical trials and subsequently assess the bias of the included studies.The Jadad scale consists of three items: (A) randomization (0-2 points), (B) blinding (0-2 points), and (C) study dropouts and withdrawals (0-1 points).For each item, there is a "yes" or "no" answer option, which results in 1 or 0 points, respectively.Studies can be given a score between 0 and 5, with a score of <3 indicating that the study is of poor quality, while studies are classified as high quality if they have a Jadad score of 3 or more.The Jadad scale is a well-known and well-applicable instrument with good reliability and validity evidence [44].
We recorded the authors, year of publication, the type of study, type or types of questionnaires for anxiety, stress, or depression assessment, the strains of microorganisms contained within the probiotic supplement, and finally, the main findings of each study.Possible discrepancies during data extraction were solved via discussion with a third investigator (EM).

Data Analysis
No statistical analysis or meta-analysis was performed due to the high heterogeneity among the studies.Thus, the data were only descriptively analyzed.

Database Searches
Overall, 16,869 records were retrieved from the database search.Duplicates were removed; hence, a total of 10,265 articles were selected.After dismissing irrelevant studies and screening the full texts of the articles, 30 studies were eligible for inclusion (Figure 1).

Studies Characteristics
Thirty publications fulfilled our inclusion criteria.Four studies focused entirely on depression, and four reported only on anxiety or stress, while twenty-two assessed both anxiety and depression scores.Concerning the type of the studies, twenty-eight were randomized controlled trials and only two were clinical trials.
The vast majority of the studies (83.3%) were of high quality.The quality scores of the included studies are summarized in Supplementary Table S1.

Microorganism Strains
Twenty-five studies utilized Lactobacillus and nineteen utilized Bifidobacterium genera, while others used a variety of microorganism genera, such as Clostridium (2), Lactococcus (3), Actobacillus (1), Streptococcus (2), and Lactiplantibacillus (1).Most of them utilized a combination of some of the aforementioned probiotics.The most used strains were as follows: Bifidobacterium longum (twelve studies), Bifidobacterium lactis (eleven studies), Lactobacillus acidophilus (ten studies), Lactobacillus casei (eight studies), and Lactobacillus helveticus (seven studies).The summary of all the strains used in each probiotic supplement can be seen on Table 1.

Assessment Tools for Anxiety and Depression
There was a variety of different kinds of assessment tools used for evaluation of anxiety and depression.The most frequently used ones were the Beck Depression Inventory (BDI) (nine studies), Depression, Anxiety, and Stress Scale (DASS) (eight studies), State-trait Anxiety Inventory (STAI), and Perceived Stress Scale (PSS) (seven studies), while Hospital Anxiety and Depression Scale (HADS) was utilized in six studies.In addition, other often used questionnaires included Hamilton Rating Scale of Depression (HAMD)-five studies, Beck Anxiety Inventory (BAI) (five studies), and the Hamilton Anxiety Rating Scale (HAMA) (four studies).Apart from that, there was also a number of 30 additional scales used, which can be seen on Table 1.

Treatment Duration
The most frequent strategy of treatment length was around 8 weeks, as was followed in eleven studies.Also, frequent treatment length was the 4-week (seven studies) and 2-week (five studies) duration.Additionally, three studies were conducted on pregnant women, thus the treatment length was equivalent to the weeks of pregnancy, while in one of these studies the treatment continued for another 6 months whilst breastfeeding.The summarized results can be seen in Table 1.

Clinical Outcomes
Most of the studies included in this review support the beneficial effects of probiotics.Kazemi et al. [46] demonstrated a significant decrease in BDI score (17.39-9.1)compared to placebo (18.18-15.55)(p = 0.042), after an 8-week use of a probiotic supplement as the main treatment, while Gawlik-Kotelnicka et al. also demonstrated clinically significant improvement in MADRS and QoL (quality of life) scores [63].On the same basis, Schaub et al. [45] showed significant improvement in depression at the 8-week follow up, with a remission rate of 55% in the probiotics group compared to a 40% in the placebo group, but only for the one subgroup with high compliance of a short-term, high-dose intake of probiotics as a supplementary treatment for depression.Regardless of the type of antidepressant used, Miyaoka et al. discovered that taking Clostridium butyricum MIYAIRI 588 in treatment-resistant depression (TRD) led to a reduction in various depression and anxiety scores by more than 50% at the end of the 8-week study [60].On the same note, Chen et al.'s study [54] supported the benefits of probiotics at 8 weeks, while Ho et al.'s [61] and Lee et al.'s [47] studies showed that the probiotic groups presented with fewer depressive symptoms and improvements in sleep.More specifically, in Lee's study those taking the NVP-1704 exhibited a greater decline in the BDI-II score compared to those on the placebo, at both the third (−6.18 ± 7.34 vs. −3.33 ± 7.03, p = 0.033) and final visits (−8.02 ± 7.17 vs. −5.39± 6.49, p = 0.036).Moreover, Yang et al.'s study [51] suggested that probiotic therapy led to reduced psychological stress before surgery, preventing patients' anxiety and heart rate from increasing.Similarly, Nikolova et al. [71] demonstrated a reduction in depressive symptoms in patients in the probiotic group with modest symptoms (HAMD-17 scores week 4: standardized effect sizes (SES), 0.70; 95% CI, 0.01-0.98,and IDS Self Report scores week 8: SES, 0.64; 95% CI, 0.03-0.87),with probiotic consumption being particularly helpful in reducing anxiety-related somatic symptoms (HAMA scores week 4: SES, 0.67; 95% CI, 0-0.95; week 8: SES, 0.79; 95% CI, 0.06-1.05).In another study, multiple sclerosis (MS) patients with depressive symptoms taking probiotic capsules for 12 weeks had significantly decreased EDSS, BDI, GHQ, and DASS scores [75], while Pinto-Sanchez et al.'s study [50] used patients with IBS and found a reduction in depression levels, but not in anxiety, when the probiotic Bifidobacterium longum NCC3001 was administered; the improvement in HAD-D scores was sustained at the 10-week follow up.
Adikari et al. [74] used EEG to investigate the effects of probiotics on physiological parameters related to anxiety in football players; after 4 weeks, they found higher delta and theta brain waves in the probiotic group, indicating, mainly that the patients were more relaxed, and their alertness was increased.Raygan et al. [70] investigated the effect of simultaneous treatment with vitamins and probiotics and showed that the intake of probiotics and vitamin D in patients with T2DM and congestive heart failure over a period of 12 weeks had positive effects on mental health parameters, including stress and depression.
Two studies utilized students as a patient group.In a study by Zhu et al. [53], the probiotic supplement prevented worsening of anxiety and significantly improved depression and sleep quality in students with mild anxiety and depression, as was also reported in another study by Venkataraman et al. [73] in students with moderate anxiety.In contrast Tran et al. [55] suggested that the beneficial effect is present only in people with severe anxiety.Similarly, Boehme et al. [66] suggested that supplementation with BL NCC3001 can improve the perception of stress.Two other studies looked at synbiotics as a treatment: Hadi et al. [72], showed that consumption of the supplement for eight weeks led to a significant improvement in stress (−3.49± 2.30 vs. −1.41± 3.44; p < 0.001), anxiety (−2.61 ± 1.49 vs. −1.46± 2.49; p = 0.03), depression (−2.86 ± 2.47 vs. −1.54± 2.13; p = 0.03) when compared to placebo.Similarly, Haghighat et al. [62] demonstrated the beneficial effects of synbiotics in depression (mean decrease in HADS-DEP score = 1.28, p = 0.009).
On the contrary, there were several studies that found subtle to no effects of probiotics on anxiety or depression.In a study by Lew et al. [68] the probiotic strain L. plantarum P8 was used in people with moderate stress, and even after 12 weeks there was no significant improvement in depression symptoms.Similarly, in Rode et al.'s study [49], although the probiotics intervention tended to lower HADS scores compared to placebo, none of the questionnaires or the Actigraphy stress ratings were associated with significant improvement due to the intervention.On the same note, Östlund-Lagerström et al. [67] and Rudzki et al. [57] also reported that the patients' symptoms did not significantly improve during the course of the treatment.Romijin et al. [58] suggested that the lack of effect of probiotics on psychological outcomes in their study was due to the length of the intervention period (8 weeks), sample size or the fact that there was no other treatments used other than probiotics.Finally, Morales-Torres et al. [59] found no significant effects and concluded that lifestyle behaviors play a role not only in mental health but also in the efficacy of probiotics; in fact, the interaction between a "good" lifestyle and probiotic intake was the only significant predictor of positive effects on anxiety, emotion regulation difficulties and mindfulness in post-treatment outcomes when controlling for gender and age.
Interestingly, in two studies, the improvement in depressive and anxiety symptoms were statistically important in both the treatment and the placebo groups [48,52].In Reininghaus' study [52] where biotin (B7) was used in combination with a multi-strain probiotic in addition to the standard treatment of depressed patients, although psychiatric symptoms improved over time in both groups, there were no differences in the outcome measures between the treatment and the placebo groups that.On the same basis, in Chahwan's study investigating the efficacy of the multispecies probiotic Ecologic Barrier in the treatment of depression, participants in both the probiotic and placebo groups showed a reduction in depressive symptoms.Analyses of one-month follow-up data revealed no significant between group differences on BDI, BAI, cognitive reactivity, and DASS scores (p > 0.05).Even after dividing the participants into groups according to severity of symptoms (mild/moderate and severe), the probiotic group did not show a greater degree of reduction in depressive symptoms.The only notable difference was found in the mild/moderate depression severity group on a test of cognitive reactivity to depressive mood, a psychological vulnerability marker suggesting that the beneficial effects of probiotics may be more noticeable in people with milder depression.
A summary of the findings in clinical outcomes can be seen in Figure 2.

Maternal Health
Three studies concentrated on maternal depression and anxiety [56, 64,65] with conflicting results.In Slykerman et al.'s study [56], pregnant women taking the probiotic Lactobacillus rhamnosusHN001 had fewer signs of postnatal anxiety and depression, compared to placebo (depression scores HN001 mean = 7.7 (SD = 5.4), placebo 9.0 (6.0), effect size −1.2, (95% CI −2.4,−0.1), p = 0.035), anxiety scores (HN001 (4.0), placebo 13.0 (4.3), effect size −1.1 −1.9, −0.2), p = 0.014).In addition, the study followed up with the women after birth and showed an association between higher levels of anxiety and depression and infant colic, which indicated that probiotic supplements could improve the mother's mood by relieving the infant's colic.After controlling for infant colic and time since birth, the reduction in anxiety (p = 0.014) and depression (p = 0.037) maintained in the long term, i.e., in the few months after birth.However, in the study by Browne et al. [65], pregnant women experienced no change in psychosocial distress after 8 weeks of probiotic consumption, and there were no differences in depressive symptomatology between the probiotic and placebo group in the 4-week postpartum follow up either.Similarly, Dawe et al. [64] found no changes in depression or anxiety in pregnant obese women between the prenatal baseline and the end of the study at week 36.

Microbiota Diversity and Abundance
Alterations in the microbiome diversity and certain genera abundance were investigated in a number of the included studies.Lee et al. [47] suggested that while the probiotic NVP-1704 showed very subtle increases in alpha and beta diversity, it induced an increase in Bifidobacterium and Lactobacillus strains and a decrease in Proteobacteria.In Schaub et al.'s study [45], the probiotics maintained microbial diversity and richness and instigated an increase in the abundance of the genus Lactobacillus after the intervention.On the same basis, Haghighat et al. [62] reported that in the synbiotic and probiotic supplemented groups, the fecal Bifidobacteria and Lactobacilicolonies numbers increased, and the coliform colonies number decreased at week 12 in comparison to the baseline.Chen et al. [54] reported that the number of genus Akkermansia increased in major depressive disorder patients after taking probiotics, while Reininghaus et al. [52], although they found no alterations in alpha diversity, demonstrated significantly altered beta diversity and a significant increase in global differential abundance of R. gauvreauii and the related Coprococcus 3. Similarly, Zhu et al. [53] demonstrated that with the use of L. plantarum, the beta diversity of the gut microbiota of patients taking probiotics was somewhat different from that of anxious students taking the placebo.
On the contrary, several studies revealed no significant alterations in the microbiome composition.In Rode et al.'s study [49], it was found that a 4-week probiotic product supplement did not alter the composition of the microbiota.Similarly, Chen et al. [54] indicated that the consumption of L. plantarum PS128 did not induce any significant changes in the microbiome.Similarly, there were no significant differences in abundance of bacterial taxa or a-b diversities in Chahwan's study [48].Meanwhile, by examining fecal 16S rRNA gene sequencing, Pinto-Sanchez et al. [50] found that the beneficial effect of BL did not depend on significant changes in microbial abundances or diversity.

Inflammation Biomarkers
Several studies measured inflammation biomarkers (TNF-a, IFNγ, IL-6) or molecules of the ACTH-cortisol signaling pathway.Venkataraman et al. [73] demonstrated that probiotics significantly reduced serum cortisol, which is associated with stress and depression, possibly by dysregulating gut barrier function and indirectly stimulating inflammatory immune responses.Although the cortisol fluctuations were very small, Lew et al. [68] found reduced levels of pro-inflammatory cytokines such as TNF-a and IFNγ in plasma, after treating with probiotics, suggesting that inflammation may have contributed to stress and anxiety.Moreover, according to the findings of Lee et al. [47], NVP-1704 lowered serum IL-6 levels and alleviated depression and anxiety attributed to the control of certain microorganisms via neuroinflammatory signaling pathways.
Chen et al. [54] also reported correlations between fluctuations in IL-6 levels and the abundance of the genus Akkermansia.Additionally, Gawlik-Kotelnicka et al. [63] reported reduced inflammation marker levels due to probiotics usage, while Miyaoka et al. [60] also suggested that C. butyricum M588 may have significant anti-inflammatory effects.Furthermore, recent data from Yang et al. [51] showed that serum corticotrophin-releasing hormone (CRF) levels and heart rate of healthy controls remained unchanged after taking probiotics suggesting that taking probiotics has no effect on the body's physiological CRF.
However, markers of the hypothalamic-pituitary-adrenal axis, including serum levels of kynurenine, tryptophan, neuropeptide Y, and ACTH-cortisol signaling pathway, were not affected by the probiotics in two studies [47,73] and four studies [49,50,57,58] demonstrated that probiotic usage did not affect any inflammation biomarker or cortisol in the subjects in neither of the groups.Finally, Haghighat et al. [62] also reported a statistically significant difference in serum brain-derived neurotrophic factor (BDNF) in the symbiotic but not in the probiotic group, while Rode et al. [49] suggests that BDNF and serotonin increased subtly, but not significantly.

Changes in Metabolic Profile
Several of the included studies measured changes in certain metabolites related to the gut microbiome, which is influenced by the probiotic usage, and their association to depression and anxiety.Rudzki et al. [57] found a significant decrease in kynurenine with the use of Lactobacillus Plantarum 299v, which was accompanied by an improved cognitive performance.In addition, Rode et al. [49] suggested that probiotics had subtle effects on markers of gut-brain interaction, such as serum serotonin concentrations, while other studies also suggested that probiotics act through serotonin pathways [72].
Moreover, Pinto-Sanchez et al.'s [50] study showed that B. Longum altered the urine metabolic profile, with methylamines and aromatic amino acids being degraded to a lesser extent by bacteria; increased 4-cresol sulfate levels were associated with lower depression scores in the B. Longum group.In addition, Zhu et al.'s study [53] showed that the probiotic treatment was linked to 13 fecal metabolomics, nearly all of which showed a negative correlation with the disrupted gut microbiome.

Discussion
The present review provides an overview of the potential clinical use of probiotics (including prebiotics/psychobiotics/synbiotics) as a therapeutic tool in the treatment of depression and anxiety.Nearly two thirds of the included studies support the modest benefit of a probiotic supplement in depression and/or anxiety (thirteen studies on depression and eleven on anxiety).Moreover, probiotic treatment appeared to be particularly helpful not only in patients with an active psychiatric illness, but also in healthy individuals experiencing stressful life events, which is in line with existing meta-analytic evidence [32].The vast majority of the studies did not report a long-term follow-up period, whilst very few described a short-term follow up of a few weeks post intervention; although no conclusions could not be drawn, due to limited data, there were no differences identified regarding anxiety/depression scores in the follow-up assessment compared to the end of intervention.It should also be noted that in nearly one third of the included studies (nine studies in total) no significant benefit was noted after the consumption of probiotics, which again is in concordance with other existing data [37,40].These conflicting findings could partly be attributable to the significant heterogeneity in the strains used in our included studies.
Interestingly, in a couple of studies, although there was improvement in the clinical picture, clinical outcomes were improved in both the treatment and the control group suggesting that preparing and consuming the probiotic daily, making appointments with the hope to improve depressive symptoms had a positive effect on mood, regardless of whether the probiotic or placebo was taken.This, along with the fact that mild depressive episodes are more likely to respond to placebo [76] and the differences noted in the mild/moderate depression severity group in one of the aforementioned studies [48], indicates that individuals experiencing milder depression may exhibit more pronounced positive outcomes from probiotics.In summary, our findings indicate that the majority of the clinical trial evidence from the last decade supports the use of probiotics in (possibly milder) depression; however, at the same time, a substantial amount of evidence shows insufficient results.
The beneficial effect on probiotic use in maternal/pregnancy mental wellbeing was also unclear; the conflicting results could be explained not only due to the small number of studies, but also due to demographic differences, dosage [77], host diet, genetics, age, medication use [78,79], and the severity of the initial symptoms, which could influence the self-reported date, as demonstrated in older studies [31].
The overall evidence in our review on the alterations induced by probiotics in gut microbiota's diversity and certain genera abundance are limited.Previous studies have shown that MDD and generalized anxiety disorder (GAD) have been associated with an increase in abundance of Proteobacteria and Enterobacteriaceae [80,81]; this is in line with one of our included studies, where probiotic use was associated with a decrease in Proteobacteria [47].Also, our review indicated that probiotic use can lead to increased abundance of Lactobacillus [45,47], species of which, such as L. casei [82], L. rhamnosus [83], and multi-strain products containing L. plantarum [53] have been implicated in modulation of psychiatric disorders and stress-induced behaviors.However, a significant proportion of the studies revealed no alterations the microbiota composition; this could partly be attributed to participants maintaining their usual dietary and lifestyle habits and the intake of the probiotic being limited to a few weeks.Findings on the alterations in alpha and beta diversity were also limited and somewhat contradictory.In summary, we were not able to detect consistent, clear evidence of specific microbiome changes following probiotic consumption.
Nearly half (thirteen) of our included studies suggested that the use of probiotics lead to decreased inflammation markers or potentially harmful metabolic molecules, however their role still needs to be elucidated.For instance, in one study probiotic consumption led to higher concentrations strains of Lactobacillus and Bifidobacterium [47], which are known to be reduced in the inflammatory state of microbiome dysbiosis.Given that neuroinflammation is an important factor in the pathophysiology of anxiety and depression [84], the effect of the probiotic treatment could be attributed to a reduction in serum proinflammatory cytokines such as IL-6.Similarly, in another of our studies, there was a link between fluctuations in IL-6 levels and the richness of the genus Akkermansia, which has been associated with proinflammatory pathways and depressive behavior in animal models [85].Results on the BDNF changes were limited and unclear.Overall, it appears that probiotic use may be associated with a reduction in neuroinflammation and the relevant inflammatory markers.However, much more consistent data is required to reach robust conclusions.

Limitations
Our review has a number of limitations.First, it is possible that the studies included in this review focused on patients with mild to moderate anxiety or depression, since patients with severe mental illness might be less able or willing to participate in clinical trials.In addition, some studies included in our review focused on specific patient groups (cancer, irritable bowel syndrome, insomnia), which may limit the generalizability of the results to all patients.Most studies were conducted on a single center and the data presented were sometimes based on a limited sample size.There was also a wide range in the probiotic supplements used and the duration of treatment, both of which may have influenced the results.Further, a general conclusion on whether probiotics were the main/only intervention or a complimentary one could not be reached, as there was no consistency in the additional psychopharmacological/psychological treatments participants were receiving, with a substantial number of studies not controlling for or not reporting this.Also, some studies included patients with mental disorders such as MDD or GAD, while other studies were conducted on healthy individuals with subclinical depressive or anxiety symptoms.In terms of the population studied, there was considerable heterogeneity, with some studies providing insufficient or no information on patient treatment and potential confounders such as duration of mental illness.Finally, several studies relied wholly or partly on self-reported data, whereas other studies included some form of clinical assessment.

Conclusions and Future Directions
The present comprehensive review discusses the most recent evidence from clinical trials of the last decade on the role of probiotic use in the treatment of anxiety and depression.Our findings indicate that in spite of some inconsistency in the results the majority of the recent literature appears to support the use of probiotics in alleviating depressive and anxiety symptoms.It appears that people with milder symptoms might benefit more from probiotic supplementation.In addition, a clear conclusion on probiotic-induced microbiome alterations could not be drawn, while a substantial amount of evidence seems to support a reduction in inflammatory markers associated with probiotics.
Probiotics could provide a non-drug-based approach in the treatment of mood disorders which could help expand the therapeutic options of both psychiatric and nonpsychiatric patients, whilst simultaneously being safe and well tolerated.In addition, as they are nutritional supplements, probiotics could help alleviate the social stigma faced by people taking psychotropic medication.Despite advances in understanding their mechanisms of action and effectiveness, a knowledge gap still exists on where and how best to use them [41].For instance, whether probiotics can be used as a complementary treatment combined with antidepressants or as a primary treatment for depression warrants further investigation, to evaluate their efficacy regardless of other medications.Current extensive research in the field of probiotics-mental health should focus on the exact mechanism of probiotics function, dosage, optimal treatment duration, and potential different outcomes depending on the severity of anxiety and depression.As there is still no clear evidence on which bacteria specifically contribute to the relief of depressive symptoms, future research should investigate different combinations of probiotics, as well as symbiotic and prebiotic effects alone, as their mechanisms of action might be different.Finally, from a methodological standpoint, given the challenges in analyzing highly heterogenous probiotic-related factors, such as interventions, populations, and outcome measures, future researchers should consider adhering to expert consensus methodological recommendations (e.g., on primary/secondary outcomes, definition of probiotic interventions, safety) [41].

Supplementary Materials:
The following supporting information can be downloaded at: https: //www.mdpi.com/article/10.3390/microorganisms12020411/s1,Supplementary Table S1: Quality assessment of included clinical trials based on Jadad scale.

Figure 2 .
Figure 2. Clinical outcomes of probiotic use in anxiety and depression.

Table 1 .
Summary of studies' characteristics.